Depression in association with neutrophil-to-lymphocyte, platelet-to-lymphocyte, and advanced lung cancer inflammation index biomarkers predicting lung cancer survival

Lung cancer is a product of inflammation and a dysfunctional immune system, and depression has similar dysregulation. Depression disproportionately affects lung cancer patients, having the highest rates of all cancers. Systemic inflammation and depression are both predictive of non-small cell lung cancer (NSCLC) survival, but the existence and extent of any co-occurrence is unknown. Studied is the association between systemic inflammation ratio (SIR) biomarker levels and patients’ depressive symptoms, with the hypothesis that depression severity would be significantly associated with prognostically poor inflammation. Newly diagnosed stage-IV non-small cell lung cancer (NSCLC; N = 186) patients were enrolled (ClinicalTrials.gov Identifier: NCT03199651) and blood draws and depression self-reports (Patient Health Questionnaire-9) were obtained. For SIRs, cell counts of neutrophils (N), lymphocytes (L), and platelets (P) were abstracted for ratio (R) calculations for NLR, PLR, and the Advanced Lung cancer Inflammation Index (ALI). Patients were followed and biomarkers were tested as predictors of 2-year overall survival (OS) to confirm their relevance. Next, multivariate linear regressions tested associations of depression with NLR, PLR, and ALI. Overall 2-year mortality was 61% (113/186). Cox model analyses confirmed higher NLR [hazard ratio (HR) = 1.91; p = 0.001] and PLR (HR = 2.08; p<0.001), along with lower ALI (HR = 0.53; p = 0.005), to be predictive of worse OS. Adjusting for covariates, depression was reliably associated with biomarker levels (p ≤ 0.02). Patients with moderate/severe depressive symptoms were 2 to 3 times more likely to have prognostically poor biomarker levels. Novel data show patients’ depressive symptoms were reliably associated with lung-relevant systemic inflammation biomarkers, all assessed at diagnosis/pretreatment. The same SIRs were found prognostic for patients’ 2-year OS. Intensive study of depression, combined with measures of cell biology and inflammation is needed to extend these findings to discover mechanisms of depression toxicity for NSCLC patients’ treatment responses and survival.


Introduction
Immunotherapy and targeted treatments are yielding significantly longer survival for patients with advanced (stage IV) non-small cell lung cancer (NSCLC) [1][2][3], but it is unknown why a substantial portion of patients fail to respond to treatment. Causes are multifactorial, but an unexplored contributor to premature death may be toxic, biobehavioral (psychological, behavioral, biological) characteristics of co-morbid depression.
Several lines of evidence point to the plausibility of depression influencing disease progression. In the general case, depression assessed at diagnosis is prognostic for mortality in cancer patients [4], with the strongest effects found in lung cancer [5]. Moreover, the continuing trajectory of depressive symptoms-from diagnosis through two years-predicts overall NSCLC survival [6]. Discovery of such effects is made possible, unfortunately, because of the high incidence of depression in NSCLC patients. Of all cancer patients, those with lung cancer are among the most impaired [5,7,8], with an estimated 36% of NSCLC with moderate to severe depressive symptoms at diagnosis, many of whom also have comorbid anxiety [9], a clinical presentation more resistant to psychological treatment [10].
Study of the health consequences of depression in lung cancer patients is timely as new therapies come on line and treatment guidelines rapidly change [23]. Psychosocial studies of lung patients have been few and come largely from prior decades of chemotherapy-only treatments [24], making examination association of depressive symptoms and systemic inflammation responses (SIRs) novel. Also important is discovery of the nature and biologic mechanisms of depressive symptom toxicity; depression is a rate limiting factor impacting patients' quality of life and, potentially, treatment response.
To do this, biomarkers of inflammation were studied in relationship to depressive symptoms. Systemic inflammatory responses (SIRs)-neutrophils, lymphocytes, monocytes, and platelets-are key factors in proliferation, angiogenesis, immunosuppression, and nutritional depletion [25]. Briefly, the inflammatory response is characterized by increases in circulating neutrophils (N) accompanied by falls in circulating lymphocytes (L). The neutrophil-to-lymphocyte ratio (NLR) is viewed as reflecting the inflammatory imbalance of pro-tumor efficacy (N) and anti-tumor capacity (L) of the host [25,26]. The platelet-to-lymphocyte ratio (PLR) is important as platelet elevation accelerates tumor progression [27,28]. Unique is the advanced lung cancer inflammation (ALI) index, which considers nutritional status with NLR. SIRs are prognostic biomarkers for multiple tumor types and multiple meta analyses have confirmed elevated NLR [29] and PLR [29] and lower ALI [29] levels at diagnosis to predict NSCLC overall survival (OS).
Newly diagnosed NSCLC patients (N = 186) enrolled in an observational cohort (NCT03199651) participated. Procedures included routine laboratory work and patientreported depressive symptom assessment [Patient Health Questionnaire-9 (PHQ-9)] [30,31]. First, NLR, PLR, and ALI were validated as predictors of OS at 2-years. If confirmed, associations of PHQ-9 scores with SIRs were next examined, with the hypothesis that depression severity would be significantly associated with higher NLR and PLR and lower ALI levels. Individually, patients with moderate to severe depressive symptoms were expected to have prognostically poor levels of systemic inflammation in comparison to patients without depressive symptoms. If confirmed, the findings would provide support for biobehavioral aspects of depression potentially "adding" to the inflammatory dysregulation already present in those with advanced NSCLC.

Study design
A prospective cohort design was used. An NCI-designated Comprehensive Cancer Center enrolled advanced (stage IV) NSCLC patients at diagnosis (NCT03199651) and followed patients up to 5 years, withdrawal, or death. Accrual occurred from July 2017 to February 2020.

Procedures
A university institutional review board approved the study and procedures in accordance with ethical standards and the 1964 Helsinki declaration. Inclusion criteria were as follows: newly diagnosed with pathology confirmed stage IV NSCLC (any type); any Eastern Cooperative Oncology Group (ECOG) performance status; any illness/condition comorbidity; age � 18 years; English-speaking; willingness to provide access to medical records, provide biospecimens, and respond to patient reported outcome (PRO) measures. Exclusion criteria were as follows: prior treatment with definitive chemo-radiotherapy for lung cancer; diagnosis >90 days prior to enrollment; receipt of lung cancer treatment for over one month; presence of disabling hearing, vision, or psychiatric conditions (e.g., psychosis) preventing consent or completion of PROs in English. Also excluded were second opinion or consult only cases (patients returning to his/her community physician for treatment).
Research personnel completed informed consent face-to-face (verbal, with written materials) during a patient's first clinic appointment with a thoracic oncologist and written informed consent was obtained and the signature witnessed. Cell count and albumin data for biomarker calculations were retrieved from electronic medical record reports. Lab data on or immediately after the patients' first visit (mean = 11.82 days) were used. Interviewers from a professional survey center contacted patients by telephone and assessed PROs, for which patients received $15. Study flow is provided (Fig 1).
Systemic inflammation biomarkers. 1) Neutrophil-to-lymphocyte ratio (NLR) was calculated by dividing a patient's absolute neutrophil count by the absolute lymphocyte count. 2) Platelet-to-lymphocyte ratio (PLR) was calculated by dividing platelet count (10 9 /L) by the absolute lymphocyte count. 3) Advanced lung cancer inflammation index (ALI) was calculated by multiplying body mass index (BMI) by the quotient of albumin (g/dL) and NLR (i.e., ALI = BMI × Albumin/NLR), where BMI = weight (kg)/height (m) 2 . Considering methods and findings from prior studies and meta analyses of SIRS as predictors of OS, the following accepted cutoffs were used for the survival analyses: 5 for NLR [32,33], 185 for PLR [34], and 24 for ALI [35].
Covariates. Variables considered were as follows: age, sex, race, partner status, education level, employment status, and smoking history. Also considered were lung cancer type, performance status, and first line treatment to be received as abstracted from medical records. The Eastern Cooperative Oncology Group Performance Status (ECOG PS) [36] score was assigned by the treating physician. ECOG PS can range from 0 to 5: 0 = fully active; 1 = restricted in physically strenuous activity but ambulatory and able to carry out light work, 2 = ambulatory and capable of all self-care, but unable to work, 3 = capable of only limited self-care, confined to bed or chair more than 50%, 4 = completely disabled, confined to bed or chair, and, 5 = deceased.

Power
A priori power analyses estimated the sample size necessary to detect a significant association of PHQ-9 scores, whether measured as continuous or categorical, with SIRs at baseline. Based on a significance level of alpha = .05, a sample size of 91 would yield an estimated power equal to 0.80 in linear regression with the six parameters and a medium effect size (Cohen's F 2 = 0.15). Sample size N = 186 translates to power >.95. This sample size is equivalent to or larger than those in meta analyses of the relationship between these biomarkers and NSCLC OS [29].

Statistical analysis
Demographic and disease characteristics of the sample were summarized and scores on depression and biomarker ratio levels and cutoffs provided. In order to avoid overfitting the survival models, covariates for analysis were selected based on a cutoff of p < 0.20 from univariate models S1 and S2 Tables. Additionally, smoking status and treatment type were included in survival models to account for their effect on survival. Demographic variables such as age, sex, race, and marital status were selected as covariates for multiple linear regressions. Baseline ECOG Performance Status was also selected to serve as a proxy for patient fitness. A cut-off of p < 0.20 was used to select additional covariates for multiple linear regressions. Cox proportional models were used to assess the association between biomarkers as defined by cutoff levels and overall survival (OS), adjusting for relevant covariates.
Next, multiple linear regressions tested the association of patient depressive symptoms (PHQ-9) with elevated NLR, elevated PLR, and lower ALI, adjusting for relevant covariates. NLR, PLR, and ALI were log-transformed due to right-skewed distributions. Descriptive analyses show the percentage of patients with no/low versus high depressive symptoms stratified by low versus high SIR levels. A p-value of 0.05 was used as a cutoff for significance. Analyses were performed using R 4.1.1. [37].
The median value for the PHQ-9 was 5.5 (Q1 = 3, Q3 = 9), corresponding to a mild level of depressive symptoms for the sample. Using classifications for individuals, 64.5% of patients reported depressive symptoms at the none/mild level (n = 120), 27.4% at the moderate level (n = 51), 4.8%% at the moderate to severe level (n = 9), and 3.2% at the severe level (n = 6).

SIR biomarkers as prognostic for NSCLC overall survival (OS)
The median follow-up for the sample was 8 months, with an inter-quartile range of 3 to 22 months and a range of 0 to 24 months. Overall mortality at 2-years was 61% (113/186). Survival analyses used the SIR cutoffs noted above and adjusted for covariates (i.e., ECOG-PS, age, educational level, smoking status, and treatment received at baseline). Analyses determined statistically significant associations between each biomarker and overall survival ( Table 2).
After adjusting for covariates, the hazard ratio (HR) for NLR is 1.91 (95% CI: 1.29, 2.84; p = 0.001). That is, patients with elevated NLR at diagnosis (higher inflammation) were approximately twice as likely to die at any time point compared to those with a lower inflammation ratio. Patients with baseline NLR<5 (47%) had an estimated one-year survival probability of 0.76 (95% CI: 0.67, 0.85), while those with NLR�5 (53%) had a one-year survival probability of 0.46 (95% CI: 0.37, 0.57). The log-rank test was significant (p-value = 0.001) indicating a marked difference in survival (Fig 2).
In summary, the relevance of the three SIRs was confirmed, with higher levels of inflammation predicting lower OS in this NSCLC sample.

Relationship of depressive symptoms with SIR biomarkers
Analyses supported the prediction of significant relationships between patients' depressive symptoms and SIRs, both obtained at diagnosis, adjusted for covariates (Table 3). That is, the PHQ-9 had a significant positive association with NLR (p = 0.02) and PLR (p = 0.02). In both cases, elevations in depressive symptoms were associated with greater elevations of neutrophils and platelets in relationship to lymphocytes. Also, PHQ-9 had an inverse, significant association with ALI (p = 0.009). High levels of depressive symptoms were associated with lower ALI values. To illustrate significant associations at the patient level, figures display the relationship between depression [PHQ-9<8 (none/mild) vs �8 (moderate to severe)] and SIRs (e.g., NLR<5 vs. NLR �5), yielding 4 patient groups determined by cutoffs. For patients with no/ mild depressive symptoms, they were as likely to be found above (50%) as they were to be below (50%) the NLR cutoff (Fig 5, left side). Although not 50/50, similar percentages of patients with no/mild depressive symptoms were found above (56%) versus below (42%) the PLR cutoff (Fig 6, left side) and below (58%) versus above (42%) the ALI cutoff (Fig 7, left  side).
In contrast, patients with moderate to severe depressive symptoms had, disproportionately, high inflammation rather than low, an effect seen across SIRs. For the depressed, 58% were above the NLR cutoff versus 42% below the cutoff (Fig 5, right side). The discrepancy is 2 to 3 times greater for PLR and ALI ratios. For the depressed, 77% were above the PLR cutoff versus 23% below (Fig 6, right side). The effect is the same, though reversed, for the depressed patients and ALI; 70% were below the cutoff verses 30% above (Fig 7, right side).

Conclusions
Systemic inflammatory biomarkers-NLR, PLR, ALI-are known predictors of advanced NSCLC survival [38]. This is supported by new data from a large sample of patients assessed at diagnosis and followed for two years. Novel data show patients' baseline depressive symptoms to be significantly associated with the same biomarker predictors, adjusting for demographic, smoking, and functional status variables. The relationship is strongest for those with moderate to severe depressive symptoms, being 1.3 to 3 times more likely to also have prognostically poor inflammation levels. Biological and psychological domains often viewed as disparate were found to covary in a significant manner, an effect replicated across the three biomarkers.
Immune-based therapies are standard of care in patients with non-small cell lung cancer [23]. A meta-analysis of eight randomized clinical trials demonstrated that the administration of chemotherapy in combination with antibodies that bind to and block the T cell PD-1 and CTLA-4 inhibitory pathways (so called checkpoint inhibitors-CPI) significantly improve overall and progression-free survival [39]. Inflammatory immune processes could alter and possibly subvert the anti-tumor immune response generated by CPI. Indeed, Ayers et al. (2021) showed elevated NLR to correlate with shorter treatment times and overall survival in lung patients receiving PD-1/PD-L1 inhibitors [33]. A large proportion of patients-35% found here-had significant depressive symptoms prior to receipt of immune-based therapy. The mechanism whereby depression leads to reduced treatment response or poorer survival is likely multi-factorial, but the current literature supports a model in which attendant inflammation exerts an inhibitory effect on immune function. These data support further investigation of depression and its mechanisms in relationship the efficacy of current therapeutic modalities.
Close inspection of the data highlighted individual differences in the association between depression and inflammation elevations. That is, data show the majority of depressed patients had prognostically poor PLR (Fig 6) and ALI (Fig 7) levels, double to triple the number of persons without depressive symptoms. The numbers are even more compelling when contrasted  with currently accepted indicators of poor survival. That is, there were more depressed/elevated inflammation patients in this study than patients with low performance status (ECOG � 2; 13%), overweight status (29%), and high school education (34%). Also, depressed NSCLC patients have a constellation of negative emotions and cognitions heightening vulnerability and lessening resilience [40]. For example, they perceive, personally, that little can be done control the disease and are pessimistic about treatment control as well. Theirs is a unique struggle with lung cancer, one different from peers, still concerned but able to cope, but doing so without a depression burden [40]. It is unknown if the depression/inflammation association would remain once cancer treatment begins. However, we have shown the trajectory of depression from diagnosis through two years (18 assessments) predicted NSCLC patients' survival (HR = 1.09, 95% CI = 1.03-1.15, p = .002), above and beyond baseline depression, sociodemographics, smoking status, cell type, and receipt of targeted treatments and immunotherapies [6]. Taken together, data support psychological, behavioral, and biologic toxicities of depression capable of influencing treatment response and/or survival.
The validity of the depression and SIR associations found here is supported by pathophysiology studies of psychiatric disorders [14,40,41] including mood disorders [15,42,43]. In NLR studies with individuals with depression but not cancer, the relationship between depression severity and NLR elevation is evident. Patients diagnosed with major depressive disorder (MDD) but not yet treated have been found with significant elevations of NLR in comparison to matched controls without MDD, an effect replicated [44][45][46]. Further, study of major depressive disorder (MDD) "severity" has found patients with MDD who previously attempted suicide had significantly higher NLRs compared to matched MDD patients without a suicide history [47]. The non-cancer studies are important for interpreting the present findings, as in combination, they show a dose response relationship between depression severity ad elevated NLR. This data provides a conceptual replication, i.e. the elevated depression/elevated inflammation effect is the same, but now observed in a cancer sample. PLR and ALI have not been studied in MDD patients, but here the depression/NLR effect was replicated in PLR and ALI analyses, adding reliability and validity the findings. And, it is likely that similar effects would be found with MDD patients. A plausible interpretation of this data is a differential effect of depression severity which "adds" to patients' basal level of inflammation arising from other sources. This may be a contributing mechanism to the uniquely high rates of depression in found LC patients at diagnosis [5,7,8] and the toxicity of the depression trajectory thereafter predicting lower survival [6].
Strengths and weaknesses are considered. Uniform timing of data collection during the difficult diagnostic period was clinically and methodologically important. The sample was at least equal to if not larger than those found in multiple SIR/OS meta analyses [27,[29][30][31][32][33]39] and sufficient for this first test of depression in relationship to lung biomarkers [48]. The SIRs are accurate, precise, and robust survival predictors across tumor types [49]. The PHQ-9 has equivalent psychometric strengths [50], and the homogeneity of the sample and the robust range of depressive symptoms found added power. Diagnostic interviews were not done, with the number of patients with MDD unknown. However, MDD criterion symptoms, e.g., low mood, anhedonia, cognitive difficulties, hopelessness, and suicidality, were endorsed by the patients, as has been the case other studies of depressed NSCLC patients [6,9,40,51]. The sample had comparatively low ethnic and racial diversity (18% vs. 22% nationally in the US) [52], potentially limiting generalizability.
In conclusion, new therapies for NSCLC are changing previously bleak, 5-month survival prognosis [53] to 2 years and longer [54]. However, variability in patient response to new therapies exists [2,3], with a pressing need to identify biomarkers predictive of first line therapy failure. The clinical significance of depression/systemic inflammation associations found here require further study as individually, the variables are prognostic for NSCLC survival [6,55,56]. Intensive study of depression among patients with NSCLC, combined with measures of cell biology, inflammation, and immunity, is needed to extend these findings and discover their mechanisms, with the long term aim to improve patients' quality of life, treatment responses, and longevity.
Supporting information S1